DESCRIPTION: This a resubmission of a proposal designed to characterize the genetic structure of selected groups of Northern Native Americans with a number of questions relating to their origin and population structure. The proposal will examine genetic variation in three linguistic groups where the proposed sampling strategy is designed to be balanced with respect to characterizing intra- and interpopulation differences; this will include two tribes of the Eskimo-Aleut, three tribes of the Na-Dene, and three tribes of the Amerid. Samples from each tribe will consist of 25 mother-father-offspring trios. This will result in approximately 75 individuals per tribe and 600 individuals overall. Genetic characterizations will focus on mitochondrial genomes, Y chromosome restriction site markers, and estimates of variation in a sample of nuclear genes including 30 microsatellite loci and four-locus haplotypes at seven well characterized autosomal regions. From this data set sequence divergence within and between populations will be estimated and characterized. A number of questions will be addressed: First, it will be possible to address the hypothesis that mtDNA lineages among the Eskimo-Aleut and Na-Dene show lower sequence diversity and thus a shorter evolutionary history than the Amerid. Second, it will be asked if the distribution of Y-associated, and thus patrilinearily transmitted haplotypes are congruent with matrilinearily inherited mtDNA lineages. This will examine the potential contribution of differential migration or fertility among males and females. The congruence of mtDNA and nuclear genes, in particular presumedly neutral microsatellite loci, will be assessed. If hypotheses concerning invasion times and population structure based on mtDNA are true then nuclear genes should show concordant features, such as reduced variation in the Eskimo-Aleut and Na-Dene compared to the Amerid. Mitochondrial sequence data will be collected by directly sequencing the non-coding control region and the NAD5 (nicotamide adenine dinucleotide dehydrogenase subunit 5) region. This involves sequencing 375 bases of the non-coding control region and the first 350-360 bases of NAD5 by established PCR-based methods using solid support biotinylated primer methods. To provide information about deep splits in the mitochondrial genealogy it is also proposed to examine informative restriction sites polymorphisms in the coding regions. These mtDNA sites have already been identified in other groups. Nuclear regions will be characterized by a variety of methods, all restriction site based from either total genomic DNA, or PCR amplified fragments. These nuclear regions are already well characterized in other diverse ethnic groups, thus providing a perspective of variation. These seven regions (HLA, ApoB, ApoAI CIII/AIV, LDLR, PAH, CFTR, and HBB) also are associated with diseases and have clinical importance. Finally a highly information rich set of microsatellite "loci" will be screened. This involves generating data on 30 loci localized to chromosomes 13 and 15 and will identify 240 alleles. A number of conventional statistical analyses are proposed to estimate allele and haplotype frequencies, test for departure from Hardy-Weinberg and examine linkage disequilibrium which can also be used in the analysis of population structure. Maximum likelihood methods such as available in Felsenstein's PHYLIP package will be used to construct molecular genealogies. These will be useful not only in reconstructing lineage affinities, but also will permit identification of admixture events. Actual tests of diversity across the hierarchical design will be tested using the AMOVA technique. Traditional tests using F- statistics will also be carried out on the nuclear polymorphisms, and Mantel test will be used to test concordance of different sets of markers. Finally, the PI proposes a series of continuing collaborations to investigate specific features of the molecular evolution of mtDNA in humans, and its ability to reflect aspects of historical demography. These include evaluating how demographic fluctuations such as population size changes and geographic subdivision influence the properties of the coalescent using both analytical and computer simulation strategies.